Throughout this application various publications are referenced by the names of the authors and the year of publication within parentheses. Full citations for these references may be found at the end of the specification immediately proceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of art as known to those skilled therein as of the date of the invention described and claimed herein.
The major species of human fibroblast interferon (IFN-.beta.) is coded for by a single gene IFN-.beta.1 (Ohno and Taniguchi, 1981; Degrave et al, 1981; Lawn et al, 1981, Gross et al, 1981; Mory et al, 1981). Expression of this gene is activated in human fibroblasts which are virus-infected or have been induced by double-stranded (ds) RNA (Burke, 1980; Raj and Pitha, 1981). After poly (rI)(rC)-induction, mRNA accumulates transiently for 2-4 hours and there is only a limited excretion of IFN unless the cells are superinduced by addition of cycloheximide or other metabolic inhibitors, which leads to a more prolonged accumulation of mRNA (Tand and Armstrong, 1970; Cavalieri et al, 1977; Sehgal et al, 1978; Raj and Pitha, 1981, 1983). In superinduced human fibroblasts, the titers of IFN-.beta. produced can reach 20-30,000 units/ml after 24 hours (Havell and Vilcek, 1972). Inducibility is a property of the human IFN-.beta.1 gene promoter, and is conserved if the gene is transferred to foreign cells by stable cotransformation (Hauser et al, 1982; Ohno and Taniguchi, 1982; Canaani and Berg, 1982) or as part of a replicating viral vector such as BPV (Zinn et al, 1982; Mitrani-Rosenbaum et al, 1983) or SV40 (Maroteaux et al, 1983a; Tavernier et al, 1983). To eliminate the need for induction and achieve a more efficient IFN production, we have prepared a cell line in which the IFN-.beta.1 gene is constitutively expressed and present in multiple copies.
The coding region of the IFN-.beta.1 gene was fused to give the SV40 early gene promoter, and then transfected into chinese hamster ovary cells deficient in dihydrofolate reductase (Urlaub and Chasin, 1980) together with a selectable DHFR gene (Kaufman and Sharp, 1982). Amplification of the fused SV40-IFN-.beta.1 DNA co-transfected with the DHFR gene, was obtained by selection of CHO transformants for resistance to increasing amounts of methotrexate (Alt et al, 1978; Ringold et al, 1981; Kaufman and Sharp, 1982). CHO cell lines excreting 200-300,000 units IFN-.beta. per ml culture in 24 hours, i.e. 10 times more than human cells and without need for induction, were obtained. The human IFN-.beta.1 produced by these hamster cells, has been purified by affinity chromatography on columns of monoclonal antibodies to native human IFN-.beta. (Novick et al, 1983) and found to have the same physical properties and specific antiviral activity as the human product.